Unraveling neuroinflammation and cytoskeleton dynamics in brain ischemia: Insights from an oxygen-glucose deprivation model of stroke in organotypic hippocampal cultures for anti-inflammatory strategies

Ischemic stroke results in neuroinflammation. While several studies addressed its molecular mechanism, we know less about the contribution of physical cues to stroke pathomechanism. In the present work, we aimed to address the significance of nervous tissue cytoskeleton rearrangements and changes in its mechanical properties in neuroinflammation. We applied oxygen-glucose deprivation (OGD) on rat organotypic hippocampal cultures (OHCs) as our model of brain ischemia. We observed that OGD significantly increases LHD and NO release from OHCs. HIF-1α protein expression was also significantly increased after OGD. We performed confocal imaging of OHCs and observed a decrease in fluorescence intensity of neuronal marker – MAP-2 and microglia marker – IBA1. IBA1-positive microglia cells possessed a lower spread area after OGD. We noticed a rearrangement of actin and microtubular cytoskeleton, which was reflected by a decrease in mean fluorescent intensity for both actin and tubulin. Finally, we perceived that OGD causes stiffening of OHCs with use of atomic force microscopy. ELISA experiment revealed a significant increase in several molecules related to inflammation (IL-6, IL-18, CCL3, CCL5, CXCL10). Thus in the next step, we tested whether chemokine receptor modulation and stromal cells conditioned media would ameliorate inflammation. We observed that using CCR2 antagonist – Irbesartan, CX3CR1 receptor antagonist – AZD8797, and dental pulp stromal cells conditioned media decreased LDH release from OGD-subjected OHCs. Summarizing our work showed that neuroinflammation resulting from OGD causes not only biological changes in OHCs but also biophysical ones. Anti-inflammatory treatment can ameliorate cell death in OHCs subjected to OGD.